The overall goal of the research is to elucidate the molecular mechanism by which coated vesicles execute sorting decisions -- how they succeed in including select sets of membrane-associated proteins during budding, how they bud, and how they fuse with the appropriate organelle after budding. An entry point into coated vesicle metabolism may have been forged in the last grant period with the identification and purification of an enzyme that can utilize ATP hydrolysis to power the removal of clathrin coating termed uncoating ATPase. We hope now to establish the exact role of this protein in the cell and its detailed mechanism of action, and to use the products of its action as substrates with which to assay for factors that may facilitate other steps in a clathrin-coated vesicle cycle of assembly and disassembly. Specifically, these and other related topics we plan to pursue in the next five years are: 1. Mechanism of ATP-Dependent Disassembly of Clathrin Coats and Its Possible in Vivo Significance 2. Reconstitution of Steps in the Clathrin-Coated Vesicle Cycle Subsequent to Uncoating 3. Functions of the Clathrin Light Chains 4. Transport of the VSV G Protein in Coated Vesicles 5. Isolation of Proteins That Bind the Carboxyterminus of VSV G Proteins, and Their Possible Role in Facilitating Transport 6. Expression of a Drosophila Heat Shock Cognate Gene in E. Coli: Does it Encode Uncoating ATPase?